Adsorption–desorption nano-aptasensors: fluorescent screening assays for ochratoxin A

In this study, a FRET-based fluorescent aptasensor for the detection of ochratoxin A (OTA) was optimized based on the quenching efficiency of single-walled carbon nanotubes (SWCNTs) and the binding affinity of aptamers. OTA aptamers were conjugated with quantum dots and adsorbed to the surface of both acid-modified and unmodified SWCNTs. The maximum fluorescence quenching efficiency of the SWCNTs were compared. Acid-modified SWCNTs (amSWCNTs) have moderate quenching efficiency, providing an optimal sensitivity for qualitative fluorescence-enhancement biosensor assays. The binding parameters of the QD-modified OTA aptamers (1.12.2 and A08min) on the surface of amSWCNTs were compared. Based on our results, the A08min aptamer is a better candidate for OTA detection. Using the A08min aptamer, the SWCNT method had a limit of detection (LOD) of 40 nM. The amSWCNT method had a significantly lower LOD of 14 nM. Turn-on fluorescent nano-aptasensors are emerging as an effective diagnostic tool for simple detection of mycotoxins. Nanocomplexes designed for the detection of mycotoxins in solution and paper-based tests have proven to be useful.


Introduction
Single-walled carbon nanotubes (SWCNTs) have stimulated multidisciplinary interest, with potential in elds such as nano electronics, 1 biology, 2-4 molecular electronics, and biomedical engineering 5,6 due to their unique mechanical, physical and chemical properties. They may also be functionalized to optimize properties for biocompatibility and biomolecular recognition. [7][8][9] Specically, one of the properties being exploited in biosensing is their quenching ability when acting as nanoquenchers and nano-scaffolds. A typical SWCNT possesses a wide absorption spectrum (approximately 500-900 nm) that overlaps with the photoluminescence spectra of various uorophores, 10,11 permitting Förster resonance energy transfer (FRET). During the energy transfer process, SWCNTs act as energy acceptors due to their delocalized p electrons, while uorophores act as donors, transferring energy to ground-state SWCNTs.
Quantum Dots (QDs), also referred to as semi conducting nanocrystals, have many advantages over traditional uorescent dyes. Among these are stronger luminescence and photostability against bleaching and physical environments such as pH, temperature, and optical turnability. Many of these properties have been exploited for immunoassays, molecular imaging, and in vivo biological labels. 12,13 In addition, QDs have also been used to facilitate the development of hybrid sensing materials. 5,14 SWCNTs can interact with QDs 15 to cause a change in their photoluminescent properties, which is useful for in vivo imaging. 16,17 Aptamers have been widely used for recognition and detection of a variety of targets in recent years. Aptamers are single stranded oligonucleotides that are selected amongst a pool of random sequences for their innate ability to recognize and bind targets with high affinity and specicity. 18 These biosensor platforms are termed aptasensors, 19 most of which rely on a conformational change in the aptamer between the bound and unbound state. This conformational change can be detected in a variety of ways, such as using uorescence, polarization, energy transfer, and colour change. These functional, single stranded DNA have been found to interact non-covalently with SWCNTs. 20,21 These complexes are stable and form by single stranded DNA (ssDNA) wrapping itself around the SWCNT through p-p stacking interactions between the nucleotide bases and the SWCNT sidewall. 20 Once the ssDNA assembles on the surface of the SWCNT, the conjugated uorophore's activity is quenched.
In the case of our sensor, ochratoxin A (OTA) is acting as the target analyte. OTA, one of the most abundant foodcontaminating mycotoxins, 22,23 is produced by fungi of the genera Aspergillus and Penicillium, which grow on a variety of crops. It is found in cereals and cereal-derived products as well as other commodities including coffee, cocoa, wine, and spices. 24 OTA is a known nephrotoxin and possible carcinogen, therefore the discovery of inexpensive, widely applicable means of detection are of substantial importance. This study will outline an effective, generally simple method for the detection of OTA with the use of unmodied SWCNT (SWCNT) and acid -modied SWCNT (amSWCNT), Quantum Dots (QDs) such as CdSe/ZnS (525 nm, green-emitting) and CdSeTe (650 nm, red emitting) and the A08min OTA aptamer (A08min). In this process, A08min-QDs act as donors, while SWCNTs act as acceptors (quenchers) (See Scheme 1 for nanocomplex 1 in the presence of amSWCNTs and S1 for nanocomplex 2 in the presence of SWCNTs). The effectiveness of the nanoaptasensors was assessed by means of uorescence, TEM, SEM imaging, and paper tests, some of which were performed under conditions mimicking eld testing.

Instruments and soware
HR-TEM images were recorded using a FEI Tecnai G2 F20 TEM, SEM images were obtained using a SEM FEG Hitachi SU-70 scanning electron microscope, UV/vis absorption spectra were obtained using a CARY 300 Bio spectrophotometer (Varian, USA) and uorescence spectra were recorded on a uorescence spectrophotometer (Horiba Jobin Yvon, USA) in the Dept. of Chemistry at Carleton University. K D values were determined for the A08min aptamer by nonlinear regression analysis of the uorescence experimental data with the one site specic binding equation using GraphPad Prism 6 soware. A highspeed Sorvall legend micro 21R (thermo electron corporation) centrifuge was used for the centrifugation of solutions.

SWCNT preparation
Unmodied SWCNTs were dissolved in MilliQ H 2 O at $1 mg mL À1 and sonicated extensively. The majority of the SWCNTs pellet out when the solution was le un-agitated for several hours; the pale-yellow supernatant was used as the unmodied SWCNT stock for trial experiments without further quantication.

Acid-modication of SWCNTs (amSWCNTs)
Acid-modied SWCNT (amSWCNTs) were prepared using the procedure developed by B. Pan and J. Liu et al. 16,25 except the mass, concentrations and ltration process were slightly modied. First, 5.2 mg of pristine SWCNTs were dissolved in 15 mL of 3.18 M HNO 3 (diluting 5 mL of conc. HNO 3 with 20 mL H 2 O) and placed in a 25 mL round bottom ask on a hot plate, equipped with a stir bar and condenser. The solution was reuxed for 27 hours and cooled for 3 days at room temperature. The resulting black solution was transferred to a 50 mL falcon tube and sonicated at room temperature for 60 min, before being reuxed for an additional 24 hours. Aer cooling, the solution was transferred back to a 50 mL falcon tube and again sonicated for 60 min at room temperature. The black solution, upon settling, separated into a yellow supernatant and a black aggregated pellet. The supernatant was passed through Spin X centrifuge lter tubes with a 0.22 mm cellulose acetate membrane (Corning) for 3 min at 2000 g. The ow-through was then passed through a 200 nm Millipore lter (1000 g, 3 min) and the amSWCNTs on the lter were washed ve times (500 mL MilliQ water, 1000 g, 3 min). The amSWCNTs on the lter paper were dried in a desiccator overnight. The dried amSWCNTs were weighed and dissolved in MilliQ water to a concentration of 0.1 mg mL À1 , then sonicated for 90 min at room temperature. Aer settling, the solution was a transparent grey-yellow supernatant with a small black pellet.

Functionalization of carboxyl QDs with amine-modied aptamers
Carboxyl CdSeTe 655 QDs and CdSe/ZnS 525 core/shell QDs were conjugated with the amine-modied aptamer (A08min) using EDC and S-NHS as cross-linking reagents. 26,27 QDs (0.93 nM, 5 mL) were mixed with 0.5 mL of EDC (25 eq. to carboxyl QDs) and 1.45 mL of S-NHS (50 eq. to carboxyl QDs) in phosphate buffered saline (PBS, pH 7.4, 450 mL). Aer shaking for 30 min, amine-modied aptamer (0.1 mM, 1.2 mL) was added and the solution was placed on a shaker at room temperature for an additional two hours. In this way, the amide linkage can form through the amino modication of the aptamer and the active carboxyl of the QDs. In order to remove the excess small molecules (EDC and S-NHS), the resulting samples were centrifuged at 14 000 rpm for 20 min.

Preparation of samples for uorescence studies
The samples of nano-complex 1 were prepared by mixing the amSWCNT (0.12 mg mL À1 , 25 mL aqueous solution), the aptamer-modied QDs (2.5 mL of 465 pM GQDs with A08min, and 2.5 mL of 465 pM RQDs with A08min) in buffer solution (10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 2.7 mM KCl, and 137 mM NaCl, pH 7.4). The various concentrations of OTA were added into the above microcentrifuge tubes. These solutions were shaken and incubated for 30 min. The remaining nanocomplex samples were prepared similarly, with the following differences. Nanocomplex 2: SWCNT (0.12 mg mL À1 , 25 mL, aqueous solution) replaced the amSWCNTs.

Preparation of OTA-spiked complex extract
The extract was prepared by mixing 2 g each of sample (wheat, barley, corn, oats and malted barley) with 50 mL of deionized water and shaking for 10 min. The mixture was centrifuged for 10 min and the supernatant was ltered through Whatman grade 1 lter paper and a syringe lter (PES 0.45 mm, 30 mm diameter). The clear solution was spiked with OTA to concentrations from 1 Â 10 À9 to 1 Â 10 À4 M (each in 1 mL).

Paper test preparation
Three rows of sample zones ($8 mm diameter circles) were prepared on unmodied Whatman grade 41 lter paper. The top row was le empty as a control lane of OTA samples. Nanocomplexes 1 and 2 (sample 1 mL) were spotted onto the bottom two rows of sample zones and le to dry for two minutes. 1 mL of 10 À4 to 10 À9 M OTA solutions in complex extract were spotted on to the top and bottom rows of sample zones. The middle row of sample zones were spotted with 1 mL of the extract alone (extract control). The paper test was then illuminated with a hand-held UV light (254 nm) and visualized with a Nikon camera (model: D7000).

TEM characterization of samples
High resolution TEM images of the A08min-amSWCNTs and A08min-SWCNTs samples were recorded by drop-casting 10 mL of a nanocomplex 1 and 2 in the absence and the presence of OTA, on a carbon-coated copper grid. Images were recorded on a FEI Tecnai G2 F20 TEM with a Schottky Field Emitter with high maximum beam current (>100 nA) electron source and imaged with a Gatan ORIUS TEM CCD Camera.

SEM characterization of samples
Scanning electron microscope (SEM) images of the A08min-amSWCNTs and A08min-SWCNTs samples were recorded by drop-casting 50 mL of a nanocomplex 1 in the absence and the presence of OTA, on a clean gold substrate and dried at room temperature. Images were obtained using a SEM FEG Hitachi SU-70 scanning electron microscope.

Results and discussion
Fluorescence screening assays for OTA-aptamers with different QDs Previously determined aptamers retaining a high affinity for OTA were compared and several aptamers were selected based on their promising binding properties for the target of interest. [28][29][30][31][32][33][34] Prior work contrasting various K D detection methods in the presence of OTA, such as microscale thermophoresis (MST), uorometric, and colorimetric assays, 28,31 suggested that the experimental parameters play an essential role in aptamer binding affinity. The uorescence screening experiments were performed with nanocomplexes using different OTA aptamers (A08min and 1.12.2) and QDs (red and green). First, we investigated the binding affinity of both aptamers modied with green QDs when incorporated into amSWCNTs with increasing concentrations of OTA. In two separate experiments, the GQD modied A08min and 1.12.2 aptamers were adsorbed on the amSWCNTs, resulting in the GQD uorescence being quenched via FRET (scheme 2A). Upon addition of OTA to nanocomplex 3, the OTA specically binds with its aptamer. As a result, the GQDs are released from the amSWCNTs and their uorescence is recovered. This shows the lower limits of detection of 37 nM for A08min aptamer and 170 nM for 1.12.2 aptamer (Fig. 1a, b and S5 †).
Similar observations were noted in nanocomplex 4 with both aptamers modied with RQDs (scheme 2B). The LOD for A08min aptamer was 50 nM and for 1.12.2 aptamer was 12.2 aptamer, 0.6 mL) and 0.12 mg mL À1 amSWCNTs. tion, we adsorbed multi-QDs (mQDs) modied aptamers (A08min-GQD and 1.12.2-RQD, Scheme 2C) to form nanocomplex 5 to give us an idea of head-to-head performance. This assay shows a LOD of 61 nM for A08min aptamer and 98 nM for 1.12.2 aptamer (Fig. 3a and b). The above experiments show that while both aptamers perform well in these experiments, the A08min aptamer produces a lower LOD when compared to 1.12.2 aptamer. The difference likely originates from the secondary and tertiary structures of the two aptamers in the bound and unbound state, as suggested from MST experiments comparing the two aptamer candidates. 28 1.12.2 is known to form a g-quadruplex in the presence of OTA. 34 The limited structural change that results from the formation of the OTA-1.12.2 complex may be responsible for the slightly elevated LOD. Thus, the A08min aptamer is a better candidate for OTA detection. The apparent K D values in these complex systems were determined for the A08min aptamer obtained using the uorescence experimental data through nonlinear regression analysis. The A08min aptamer apparent K D s were different in different environments, which was anticipated as there would be variable affects of the interaction of the aptamer with the surface that could impact the measured affinity. The aptamer binds with high affinity (nanomolar range from 18-54 nM) in solutions containing amSWCNT (Fig. S10 †). Moreover, these data are in good agreement with our previous reported analysis. The microscale thermophoresis (MST) showed K D ¼ 97 AE 33 nM, the uorometric SYBR Green assay showed K D ¼ 169 AE 52 nM and affinity chromatography assay (magnetic beads) showed K D ¼ 406 AE 166 nM. 31 These results conrm that the A08min aptamer binds OTA with excellent affinity and therefore may be the best aptamer for our OTA assays.

Fluorescence solution studies
With the optimal OTA aptamer selected, we retained the concept of dual color detection (employing both the GQD and RQD labels with the A08 aptamer) for two reasons. Firstly, having two signals to monitor could allow for an internal check to conrm binding results. Secondly, this format could be adapted to multiplexing in future experiments. The QDs employed for this work have two strong emission wavelengths, one at 525 nm (GQD) corresponding to CdSe/ZnS and the other at 650 nm (RQD) corresponding to CdSeTe (Fig. S3a †). The emission spectra of GQD and RQD overlap with the absorption of amSWCNTs (Fig. S3b †) allow FRET to occur, resulting in QDs uorescence quenching. The emission spectra of A08min-QDs in the presence of different concentrations of SWCNT and amSWCNTs were investigated. As shown in Fig. S4a and b, † the uorescence intensities of A08min-QDs decrease while the concentration of amSWCNTs ranging from 0.0 to 0.12 mg mL À1 , was increased. The maximum quenching was observed at an amSWCNT concentration of 0.12 mg mL À1 . The GQD uorescence was quenched to 37% of maximum. Similarly, the RQD uorescence was quenched to 13% of maximum. The interaction holding the A08min-QDs to the amSWCNTs is noncovalent and yields nanocomplex 1. It forms through the p-p stacking/van der Waals forces of the nucleotide bases to the amSWCNT sidewall, [35][36][37] bringing the QDs in close proximity to the amSWCNT. The energy transfer occurs from the aptamer-QDs, which act as donors, to the amSWCNTs, which act as acceptors, resulting in the uorescence quenching of A08min-QDs. The maximum uorescence quenching efficiency, observed when 0.12 mg mL À1 of each amSWCNT was added, was greater than 65% for amSWCNT, and 99% for SWCNT (nanocomplex 2). In a recent study of the noncovalent interactions of SWCNTs in the presence of uorescein derivatives, quenching efficiency was between 67% and 98%. 38,39 This limited quenching efficiency of amSWCNTs provides sufficient sensitivity for uorescence enhancement qualitative biosensor assays.
With the addition of OTA (concentration range of 0 M-1.2 mM) to nanocomplex 1, OTA binds specically with its aptamer, leading to a conformational change of the A08min aptamer. This conformational change results in the disruption of the p-p stacking interactions between A08min-QD and the amSWCNT, thus causing it to release from the amSWCNT surface. As a result, FRET is inhibited and the uorescence of A08min-QDs is recovered (Scheme 1 and Fig. 4a). This assay showed good linearity in the range from 0 M to 160 nM OTA with a limit of detection (using 3.3 Â Syx/slope) of 14 nM (Fig. 4b). Similar experiments were performed for OTA with nanocomplex 2. The experiments were carried out using the same concentrations and conditions listed above which were used to perform the nanocomplex 1 method. Similar observations were noted to those for detection of OTA with nanocomplex 1. This uorescence assay showed good linearity in the range from 0 M to 160 nM OTA with a limit of detection (using 3.3 Â Syx/slope) of 40 nM (Fig. 5a, b and S1 †). The selectivity experiments for OTA over other mycotoxin standards such as Warfarin, AFB1, DON, Patulin, FB1 and OTB were added to the nanocomplexes under the same experimental conditions. The results demonstrated that no signicant change in the uorescence intensity occurred (Fig. 6). These observations indicate that nanocomplex 1 is selective for OTA.

HR TEM studies
To conrm our hypothesis that the displacement of the aptamer from the SWCNT surface is what is leading to our signal enhancement, TEM samples were prepared by drop casting the solutions onto a carbon coated copper grid. Bundles of amSWCNT that are 40-100 nm in diameter and amSWCNT bundles at 20-50 nm in diameter could be observed by TEM. CdSe/ZnS and CdSeTe QDs were also imaged; the average diameter of each was 3.5 nm and 5.0 nm respectively (Fig. S2 †). The proposed mechanism for the assembly and disassembly of nanocomplex 1 and nanocomplex 2 was supported by TEM analysis. Fig. 7a-c shows A08min-QDs (dark particles) on the outer surface of amSWCNT (tube shaped structure). This indicates the A08min-QDs were adsorbed onto the amSWCNT through the noncovalent p-p stacking interactions to form nanocomplex 1. Fig. 7d-f shows the separation of A08min-QDs from the amSWCNT upon addition of OTA. This indicates that A08min-QD preferably binds OTA over the amSWCNT. The same experimental conditions were used for nanocomplex 2 and similar results were observed. TEM images of nanocomplex 2 in the absence and the presence of OTA are illustrated in Fig. 8. Nanocomplexes 1 and 2 assembly and disassembly were also observed throughout the sample area in the grid, and the images are from multiple regions as presented in ESI (Fig S7 and S8) †.

SEM studies
Nanocomplex 1 was also characterized by SEM. SEM samples were prepared by solvent evaporation on a gold planar surface. All of the SEM studies discussed follow the same experimental conditions as the previous TEM study. The images of nanocomplex 1 illustrate assembly in the absence, and disassembly in the presence of OTA. Fig. 9a shows nanocomplex 1 in the absence of OTA, where the A08min-QDs adsorb to the surface of  the amSWCNT. Upon the addition of OTA, OTA specic binding with its aptamer results in the disassembly of the A08min-QDs and amSWCNTs from nanocomplex 1. This is depicted in Fig. 9b where the A08min-QDs and amSWCNTs are welldispersed.

OTA detection in extracts
Additionally, other tests were performed using OTA-spiked extracts to test the effectiveness of this solution-based sensing. These extracts were a mixture of wheat, barley, corn, oat and malted barley to simulate authentic testing conditions. QD uorescence was recovered to roughly 55% of the original signal intensity, slightly less than nanocomplex 1 in buffer demonstrated in the previous experiments (Fig. 10a). This assay showed good linearity in the range from 0 M to 160 nM OTA with a limit of detection (using 3.3 Â Syx/slope) of 0.018 mg g À1 (Fig. 10b). This slight difference in quenching effect is likely due to other components of the extract contributing as quenchers to the QDs. Still, the nanocomplex 1 approach was deemed feasible for mycotoxin detection in both simple and complex matrices.
Furthermore, paper-based detection systems were developed based on the approach of nanocomplex 1. Paper assays are gaining widespread attention in biosensor development due to their low cost and simplicity. DNA-nanoparticle paper-based assays have been developed for the detection of nucleic acid and non-nucleic-acid targets. [40][41][42] To set up the paper-based test, Fig. 10 (a) The fluorescence spectra of nanocomplex 1 with OTA-spiked extracts. After assembly of the nanocomplex 1 with A08 aptamer (red line), OTA-induced disassembly leads to an increase in the fluorescence signals (blue lines), to 55% of the original signal prior to assembly (dashed line). The loss of fluorescence in comparison to buffer solution is attributed to matrix effects. (b) Relative fluorescence at 532 nm (blue dots, y ¼ 6.1863x + 1.7737, R 2 ¼ 0.8671) and 630 nm (red dots, y ¼ 1.9045x + 0.2789, R 2 ¼ 0.9089) versus OTA concentrations. Inset: shows linear dynamic range. (c) 1 mL OTA from 10 À4 to 10 À9 M was spiked into complex extract and spotted into top row as a control. Nanocomplex 1 sample (1 mL) was spotted into middle row as a control. Both OTA-spiked extracts from 10 À4 to 10 À9 M and nanocomplex 1 were spotted onto the bottom row (Table 1).
rst OTA spiked extracts were dabbed onto the top and bottom rows of the lter paper at concentrations ranging from 10 pg g À1 to 10 mg g À1 . The top row was used as a control, while nanocomplex 1 was added to the bottom row. Nanocomplex 1 was also spotted on the middle row as a control. When compared to controls, visual detection of OTA, down to 100 ng g À1 , could be observed with a simple hand-held UV-light and camera, without paper modication or the need for expensive equipment (Fig. 10c). When attempting the paper-based test for extracts with nanocomplex 2, using similar experimental conditions described previously, the results were less successful. Only in the most concentrated trial, at a concentration of 100 mM, was any uorescence established (Fig. S9 †). This clearly establishes the superiority of nanocomplex 1 for the sensing of OTA in extracts as the presence of amSWCNTs led to the 100-fold increase in sensitivity over nanocomplex 2.
For comparison purposes, we reviewed several FRET-based aptasensors for OTA which use different recognition materials such as Graphene oxide (GO) and SWCNTs (Table 1). These methods were less sensitive, having LODs from 17.2 nM-24.1 nM 37-39,41-44 compared to our FRET method which had LOD of 14 nM. In addition, our method has several advantages over FRET-based OTA sensors. It is relatively fast, requiring only 30 min to perform the experiment. Additionally, we compared the sensitivity and selectivity for both unmodied and modied SWCNTs and two different OTA aptamers labeled with two different QDs for easy uorescence interpretation. In these regards, our method is better than other detection methods. Our proposed amSWCNT aptasensor was simple, providing better reproducibility and convenient observations within 30 min. It was successfully applied in paper tests and for real sample analysis. This method is promising for future on-site mycotoxin testing applications.

Conclusions
The results conrm that nanocomplex 1 effectively translates the binding of the aptamer to OTA into a specic biosensor. This binding was used as a switch, turning on the uorescence in the presence of OTA. This approach proved to be effective in both paper and solution-based tests, even when the target was introduced in agricultural extracts. Multi QDs (mQDs) were used to emit different wavelengths of light upon excitation with a UV lamp. Future applications may manipulate this property to create a multi-toxin biosensor by conjugating different coloured QD to aptamers selected for different toxins.

Conflicts of interest
There are no conicts to declare.